Interactome analysis of Bag-1 isoforms reveals novel interaction partners in endoplasmic reticulum-associated degradation

Abstract

Bag-1 is a multifunctional protein that regulates Hsp70 chaperone activity, apoptosis, and proliferation. The three major Bag-1 isoforms have different subcellular localizations and partly non-overlapping functions. To identify the detailed interaction network of each isoform, we utilized mass spectrometry-based proteomics and found that interactomes of Bag-1 isoforms contained many common proteins, with variations in their abundances. Bag-1 interactomes were enriched with proteins involved in protein processing and degradation pathways. Novel interaction partners included VCP/p97; a transitional ER ATPase, Rad23B; a shuttling factor for ubiquitinated proteins, proteasome components, and ER-resident proteins, suggesting a role for Bag-1 also in ER-associated protein degradation (ERAD). Bag-1 pull-down from cells and tissues from breast cancer patients validated these interactions and showed cancer-related prominence. Using in silico predictions we detected hotspot residues of Bag-1. Mutations of these residues caused loss of binding to protein quality control elements and impaired proteasomal activity in MCF-7 cells. Following CD147 glycosylation pattern, we showed that Bag-1 downregulated VCP/p97-dependent ERAD. Overall, our data extends the interaction map of Bag-1, and broadens its role in protein homeostasis. Targeting the interaction surfaces revealed in this study might be an effective strategy in the treatment of cancer.

Valosin containing protein (VCP): initiator, modifier, and potential drug target for neurodegenerative diseases

“…Bag-1 enables the formation of a ternary CHIP/Bag-1/VCP complex [ 165 ]. This may involve two Bag-1 binding sites in the D1 domain of VCP [ 70 ]. The importance of the Bag-1/VCP interaction goes beyond the removal of SOD1 G93A .…”

“…The importance of the Bag-1/VCP interaction goes beyond the removal of SOD1 G93A . Bag-1 also regulates ERAD, at least for some VCP clients [ 70 ]. Taken together, CHIP and Bag-1 illustrate the intricate connections between VCP and other pillars of the intracellular proteostasis network.…”

Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70

“…Bcl-2-associated anthanogene 1 (Bag-1) is a multifunctional, prosurvival protein that binds to Bcl-2 and promotes cell survival [ 13 ]. However, the predominant function of Bag-1 was reported to be a co-chaperone for the 70 kDa heat shock protein (HSP70) to activate and regulate its protective function [ 14 , 15 , 16 ]. The Bag-1 gene encodes three major protein isoforms in human cells: Bag-1L (52 kDa), Bag-1M (46–48 kDa) and Bag-1S (36 kDa) in human cells, and 32 kDa (Bag-1S) and 50 kDa (Bag-1L) isoforms in mouse cells [ 17 , 18 ].…”